- Email: [email protected]
The electrophoretic homogeneity of the myosin subunits
462
R. E. WINNICK, H. LIS, T. WINNICK ACKNOWLEDGEMENTS
We thank Dr. MONTAVU of Hoffman-La-Roche, Basel (Switzerland), for a supply of 5-fluorouracil, and Professor E. D. BERGMANN Of the Hebrew University, Jerusalem, for a sample of 5-fluorotryptophan. REFERENCES 1 G. V. CAUSE AND M. G. BRAZHNIKOVA, Lancet, (1944) 715 . 2 R. SCHWYZER, Record Chem. -Progr. (Kresge-Hooker Sci. Lib.), 20 (1959) 147. 3 j . M. BARRY AND E. ICHIHARA, Nature, 181 (1958) 1274. 4 j. L. STOKES AND C. R. WOODWARD, J. Bacteriol., 46 (1943) 83. s B. D. DAvIs AND E. S. MINGIOLI, J. Bacteriol., 60 (195o) 17. s E. KATZ, J. Biol. Chem., 235 (196o) lO9O. 7 W. D. MCELROY AND H. B. GLASS, A symposium on amino acid metabolism, J o h n s H o p k i n s U n i v e r s i t y Press, Baltimore, 1955, p. 277. s A. MEISTER, Biochemistry ol the amino acids, Academic Press, New York, 1957, C h a p t e r 4. 9 L. GORINI AND W. K. MAAS, Biochim. Biophys. Acta, 25 (1957) 2o8. 10 F. ZILLIKEI~!,Federation Proc., 18 (1959) 966. 11 V. V. KONINGSBERGER, C. O. VAN DER GRINTEN AND J. T. G. OVERBEEK, Biochim. Biophys. Acta, 26 (1957) 483 • 12 j . M. KIRK, Biochim. Biophys. Acta, 42 (196o) 167. 13 D. H. STRUMEYER AND K. BLOCH, J . Biol. Chem., 235 (196o) PC 27. 14 H. M. BATES AND F. LIPMANN, J. Biol. Chem., 235 (196o) PC 22. is E. ITo AND J. L. STROMINGER, dr. Biol. Chem., 235 (196o) PC 5. is :R. HANCOCK, Bioehim. Biophys. Acta, 37 (196o) 4717 I. UEMUZA, I~. SUYAMA AND C. J. CHERN, J . Osaka City Med. Center, 9 (196o) 1321, 2643, 2861.
Biochim. Biophys. Acta, 49 (1961) 451-462
THE ELECTROPHORETIC
HOMOGENEITY
OF T H E M Y O S I N S U B U N I T S PARKER
A. SMALL*, W I L L I A M F. H A R R I N G T O N * * AND W. W A Y N E K I E L L E Y
Laboratory o] Cellular Physiology and Metabolism, National Heart Institute, National Institutes oI Health, Bethesda, Md. (U.S.A .) Received N o v e m b e r 3rd, 196o)
SUMMARY
Gel electrophoresis of myosin in concentrated urea solutions demonstrates that the dissociated chains of myosin migrate as a monodisperse electrochemical species. At lower urea concentrations a complex dissociation-association system is obtained. Sedimentation, viscosity, optical rotation and gel electrophoresis studies under the latter conditions indicate the equilibrium nature of the system. Abbreviations: D M A P N , 2-dimethylaminopropionitrile; NEM, N-ethylmaleimide; Tris, tris(hydroxymethyl) aminomethane. * At p r e s e n t w i t h the W r i g h t - F l e m i n g I n s t i t u t e , St. M a r y ' s H o s p i t a l Medical School, Paddington, L o n d o n (Great Britain). ** P r e s e n t address: McCollum P r a t t I n s t i t u t e , J o h n s H o p k i n s University, Baltimore, Md. (U.S.A.).
Biochim. Biophys. ,4cta, 40 (1961) 462 -47 o
ELECTROPHORESIS OF MYOSIN IN UREA
463
INTRODUCTION
In a recent report t evidence was presented demonstrating that the myosin molecule is dissociated in aqueous 5 M guanidine. HC1 into three polypeptide chains of similar, if not identical, mass. The present study is concerned with the possible identity of the three polypeptide subunits. As a first approach to this problem, myosin-urea systems have been subjected to electrophoresis, utilizing an acrylamide gel (cyanogum No. 41 ) as matrix. As we will demonstrate below, urea concentrations of the order of 12 M are required to maintain complete dissociation of the individual chains of myosin. Under these conditions the polypeptide chains migrate as a single relatively sharp band in gel electrophoresis experiments over the p H range 7.2-9.5. I t thus appears that the subunit chains of myosin are electrochemically identical.
Myosin
MATERIALS AND METHODS
The method of preparation of myosin for these studies has been fully described elsewhere 1.
Experimental methods Myosin concentrations were determined spectrophotometrically assuming -279~I%_-5.60, a value recently established b y GELLERT AND ENGLANDER~ from careful dry weight measurements*.
Sedimentation studies Sedimentation was observed in a Spinco Model E Ultracentrifuge operating at 59,780 rev./min. Temperature control was achieved through the RTIC unit of the instrument. All runs were made in analytical cells equipped with Kel-F. Centerpieces. Sedimentation coefficients were corrected to a standard state of water at 20 ° assuming negligible binding of urea b y the protein (see ref. I for discussion); viscosities and densities of the various aqueous urea solvent systems were measured at the temperature of the ultracentrifuge runs ( + I°). In the calculation of s20,w values, a partial specific volume, ~ = 0.720 ml/g, was assumed. This was the value determined for myosin in 5 M guanidine.HC1 (see ref. I) and is consistent with the observations of KAY4 who showed t h a t the partial specific volume of a number of proteins decreases about 1 % when they are transferred to concentrated aqueous urea solvent.
Gel electrophoresis A modification of the gel electrophoresis method of RAYMOND5,6 was employed utilizing gels of acrylamide. These were formed in the presence of a suitable buffer system and the appropriate concentration of urea. The mixture of acrylamide and N,N' bismethyleneacrylamide monomer and the DMAPN were generously donated b y the American Cyanamide Company and are normally available under the trade names of Cyanogum No. 41 monomer and DMAPN catalyst, respectively. 3 g of * I n a n e a r l i e r p u b l i c a t i o n f r o m t h i s l a b o r a t o r y I t h e 2nd v i r i a l coefficient for m y o s i n i n 0. 5 M KC1 w a s g i v e n as 0.9o" IO-¢ mole m l / g z. T h i s n e w v a l u e for a b s o r b a n c y a t 279 m # ( c o n c n . = i %) c h a n g e s t h e v a l u e for t h e 2rid v i r i a l coefficient t o o.75" IO-e mol e m l / g I b u t does not, of course, a l t e r t h e i n f i n i t e d i l u t i o n v a l u e for t h e m o l e c u l a r w e i g h t (619,ooo). F o r t h e s a m e r e a s o n our p r e v i o u s l y p u b l i s h e d figure for t h e SH c o n t e n t of m y o s i n b e c o m e s 7.4 m o l e s / l ° s g.
Biochim. Biophys. Acta, 49 (1961) 462-47 o
464
P. A. S M A L L , W. F. H A R R I N G T O N ,
W. W. KIELLEY
monomer, the appropriate weight of urea and the volume of buffer solution noted in Table I, were diluted to a volume of 80 ml with water. After filtering the solution, 0.32 ml DMAPN catalyst and o.32 ml of a freshly prepared IO % ammonium persulfate solution were added (in that order) to initiate polymerization. Solutions were stirred gently to achieve mixing without aeration, then transferred to a lucite electrophoresis t r a y (25 × 4 × 0.5 cm) and covered. At room temperature, gelation required 30 to 12o min; at 40-50 ° (the temperature of the electrophoresis runs in 12 M urea system) gelation occurred in 3-30 min. TABLE
I
BUFFER SYSTEMS FOR GEL ELECTROPHORESIS pH
Gel (final v o l u m e 8 0 ml) Bridge (final v o l u m e 5 0 0 m l )
Stock reagents
7.z
8.6
9.5
2.o M Tris i.o M citric acid
2. 7 m l 2.0 m l
3.04 ml 0.40 ml
5.0 ml 0.05 ml
0. 5 M b o r i c a c i d 2. 5 M N a O H
5° ml o.I ml
3° 2. 4
ml ml
3° 4
ml ml
In general, the procedure of SMITHIES7 was followed in making electrophoresis runs. The protein, of concentration 5-25 mg/ml, was applied to a strip of W h a t m a n No. I filter paper which was then inserted into the gel (prior addition of urea to the myosin solutions was found to be unnecessary). A few drops of mineral oil were layered over the acrylamide matrix and the t r a y was covered with Saran Wrap to prevent evaporation of moisture during electrophoresis. The bridge buffer systems were made according to Table I. A voltage drop of 3-6 V/cm was sufficient to achieve an adequate migration rate. After completion of a run, electrophoretic patterns were developed b y placing the gel in a 1 % solution of Amido Black lOt3 dye (Hartman Leddon Company, Philadelphia) in methanol-water-acetic acid (5:5 : I, v/v) for 5-1o rain. Excess free dye was removed by washing the gel in m a n y changes of the same solvent (5:5:1) over a period of 4-5 days. RESULTS AND DISCUSSION
The dissociation of the myosin subunits in urea S N E L L M A N AND ERDOSs have demonstrated that myosin in aqueous 6 M urea solvent exhibits a complex sedimentation pattern. This is in contrast to sedimentation studies on myosin in aqueous 5 M guanidine. HC1 where the protein material sediments as a single weight class at all protein concentrations as demonstrated in Fig. I. A small, slow moving component, magnified b y the JOHNSTON-OGSTON affect, is apparent in the double sector cell experiment (6.1 mg protein/ml) in Fig. I. However, experience in this laboratory indicates that this material is a contaminant and can be considerably reduced b y repeating the ammonium sulfate fractionation in the preparation of myosin. All of our previous work on the dissociation of myosin into its component subunit chains was effected in 5 M guanidine. HC1, which is obviously unsuited for electrophoretic experiments. Consequently, a number of sedimentation B i o c h i m . B i o p h y s . A c t a , 4 9 (1961) 4 6 2 - 4 7 °
ELECTROPHORESIS OF MYOSIN IN UREA
465
Fig. I. Velocity s e d i m e n t a t i o n of m y o s i n in 5 M guanidine.HC1. The 2 and 4 m g / m l samples were t r e a t e d w i t h N E M and centrifuged at 59,78o r e v . / m i n for 179 a n d 189 rain, respectively; t h e 6.1 m g ] m l s a m p l e w a s u n t r e a t e d a n d centrifuged in a double sector cell at 5o,74 o r e v . / m i n for 448 m i n ; the 12. I m g / m l s a m p l e was treated with N E M and centrifuged f r o m a s y n t h e t i c b o u n d a r y ; the picture was t a k e n 128 rain after the centrifuge was b r o u g h t to 59,780 r e v . / m i n following I2o min at 12,34o.
experiments on various solutions of myosin in aqueous urea were performed in order to establish conditions for complete dissociation of the native molecule in this solvent*. Fig. 2 presents ultracentrifuge patterns obtained at various urea concentrations where it m a y be seen that complete dissociation of the myosin molecule into the slowest moving ( s z o , w ~ 1-2 S) peak is achieved only at urea concentrations of the order of 12 M (40°). The myosin samples employed in the experiments summarized in Fig. 2 were pretreated with NEM to prevent disulfide bridging among the dissociated chains 1. However, similar results were obtained when native myosin was added directly to either concentrated aqueous urea solvents or aqueous urea in the presence of o.I M fl-mercaptoethanol. In these experiments the ionic strength of the solvent system was
Fig. 2. Velocity s e d i m e n t a t i o n of m y o s i n as a f u n c t i o n of urea concentration. All p r o t e i n concent r a t i o n s 8 m g / m l ; KC1 concentration, 4-o.12 M, except the 2 M urea e x p e r i m e n t where 0.27 M KC1 was present. T e m p e r a t u r e , 2o-25 ° except for the 12 M urea e x p e r i m e n t (43% F o r o t h e r conditions, see text. * We are i n d e b t e d to Drs. J. T. EDSALL and D. ]3. WETI_AtIFER for informing us of their sedim e n t a t i o n w o r k on m y o s i n in 8 M urea solutions prior to publication.
Biochim. Biophys. Acta, 49 (1961) 462-470
466
P . A . SMALL, W. F. HARRINGTON, W. W. KIELLEY
critical. Thus, when myosin in IO M urea-o.I2 M KC1 solvent was examined in the ultracentrifuge, most of the protein sedimented as a slow moving boundary with sz0, w ---- 1.7 S (at a concentration of 8. 3 mg/ml) as shown in Fig. 2. On the other hand, myosin in IO M urea and the electrophoresis buffer system (pH 8.2) is predominantly undissociated. The sedimentation versus concentration dependence of myosin in various urea concentrations is shown in Fig. 4. In 12 M urea solvent where a single sedimenting boundary is observed (Fig. 2) the concentration dependence of sedimentation is much lower than that found for the major peak at lower urea concentrations. A plot of I/S versus C is linear and m a y be fitted b y the equation: I
I -
s
sO
(i + kc)
where c is the concentration in g/ml, k ---- 178 ml/g and S o ~- 3.51 S, the value of szo, w at infinite dilution. Assuming dissociation of the myosin macromolecule into random polypeptide chains of a single weight class in 12 M urea, it was of interest to estimate the chain weight. A molecular weight oi 18o,ooo was obtained from the FLORY-MANDELKERN equation 9, and the parameters s20' w ~ 3.51 S (Fig. 4) and E~] ---- 1.17 dl/g (Fig. 5). This value should be compared to a value of 197,ooo found in 5 M guanidine. HC1 (see ref. I) and supports our view that the chains are completely dissociated in 12 M urea.
Electrophoretic properties of the dissociated chains o/myosin Electrophoresis patterns of myosin in 12 M urea at various apparent p H values are shown in Fig. 3, demonstrating that a monodisperse migrating band was obtained under these solvent conditions over the p H range 7.2-9.5. At p H 8.6 and pH 9.5 the band had moved over essentially the length of the cyanogum gel with no indication of splitting. At p H 7.2 the migration rate was extremely slow, and in the time employed (48 h) for this study, diffusion had widened the band somewhat. At p H 9.5 electrophoresis for shorter time periods (5 h) yielded a single sharp band in contrast to the somewhat diffuse band depicted in Fig. 3 at this pH.
Fig. 3. Acrylamide gel electrophoresis of m y o s i n in 12 M urea a t different a p p a r e n t p H values. H o r i z o n t a l a r r o w indicates direction of migration, vertical arrows indicate origins. F o r conditions, see text.
The mechanism o/the dissociation o[ myosin in urea The complex behavior patterns of myosin in the presence of urea concentrations lower than 12 M is evident in Fig. 2. Several sedimenting components are present B i o c h i m . B i o p h y s . A c t a , 49 (i96I) 462-47 °
467
E L E C T R O P H O R E S I S OF MYOSIN IN U R E A
in any given urea concentration. A slow moving component is evident at urea concentrations of 2-8 M. This may be the contaminant referred to earlier. The sedimentation versus concentration dependence of the major peak (see Fig. 2) at various urea concentrations is presented in Fig. 4. In the 2 and 4 M urea systems it is apparent that very large molecular weight aggregates exist in the measurable concentration range of protein exhibiting infinite dilution values of S~o, w greater than 20 S. Higher urea concentrations favor the dispersal of these aggregates into the monomeric polypeptide chains. Viscosity measurements on the various urea solutions also emphasize the aggregation phenomena. Fig. 5 presents a system of reduced viscosity versus concentration plots at various urea concentrations. In general, data were obtained 24-48 h after the addition of urea or after the outflow times had reached constant values. Measured viscosities were essentially unchanged by the absence or presence of fl-mercaptoethanol. The extreme slope of the ~tsp/C versus C plot and the high values of ~sp/C at finite protein concentrations in 2 M urea is evidence of the massive concentrationdependent association in this solvent. One aspect of Fig. 5 is particularly striking. This is the apparent discontinuity in the sequence of the reduced viscosity versus concentration plots at 4 M urea where ~tsp/C versus C curve abruptly falls to comparatively low values. Nevertheless, the slope of the plot in 4 M urea suggests the presence of concentration-dependent aggregate, a view which is supported by the magnitude of s20,w of the major component observed in sedimentation studies (see Fig. 4). 7.0 6.5
20
x~
I
I
I
I
r
I
I
6.O
5.5 5.0
16
4.5
t4
12
~
4,0
~
3,5
o
4U
~'/"
3.(1 8 6 4
2 0
I
I
I
2
I' 3
I
I
I
I
I
4
5
6
7
S
PROTEIN CONCENTRATION (m~l/ml)
Fig. 4. C o n c e n t r a t i o n d e p e n d e n c e of s e d i m e n t a t i o n c o e f f i c i e n t s , s 20, w. a s a f u n c t i o n of u r e a c o n c e n t r a t i o n . 2 M u r e a , O; 4 M u r e a , & ; 8 M urea, [] ; i o M u r e a , • ; 12 M u r e a , ~ . T h e d a s h l i n e i n d i c a t e s t h e b e h a v i o r of n a t i v e m y o s i n in o. 5 M IZC1. F o r o t h e r c o n d i t i o n s , see t e x t .
0
0.1
0.2
0.3
0.4
o.5
0.6
0.7
0.8
PROTEIN CONCENTRATION ( g / l O O m | )
Fig. 5. R e d u c e d v i s c o s i t y v s . p r o t e i n c o n c e n t r a tion at various urea concentrations. 2 M urea, O; 4 M u r e a , &; 8 Murea, []; ioMurea, •; 12 M u r e a , <~. D a s h l i n e i n d i c a t e s b e h a v i o r of n a t i v e m y o s i n i n o. 5 M KC1.
It is instructive to compare changes in the optical rotatory properties which are a reflection, primarily, of the unfolding of the individual chains, with the viscosity Biochim. Biophys. A cta, 49 (1961) 4 6 2 - 4 7 °
468
P.A.
SMALL,
W. F. H A R R I N G T O N ,
W. W. KIELLEY
a n d s e d i m e n t a t i o n p r o p e r t i e s which sense the gross size a n d shape of t h e m a c r o m o lecules. I n 2 M urea, t h e specific o p t i c a l r o t a t i o n levels off a t a value (see T a b l e II) which is a b o u t 25 ~o of t h e over-all r o t a t o r y change o b s e r v e d a t high u r e a c o n c e n t r a tions. Y e t a t 4 M u r e a t h e m y o s i n chains are a b o u t 9 ° °/o u n f o l d e d as r e v e a l e d b y t h e values of [~]D a n d 2e, t h e D r u d e p a r a m e t e r , in T a b l e I I . Thus, it would seem t h a t in 2 M u r e a t h e m y o s i n molecules r e t a i n a considerable degree of r i g i d i t y which is conferred b y t h e presence of t h e high s-helical c o n t e n t , whereas a t 4 M u r e a t h e individual chains are e s s e n t i a l l y u n f o l d e d leading to a collapse of t h e rod-like s t r u c t u r e a n d t h e s u d d e n decline in r e d u c e d viscosity o b s e r v e d in Fig. 5. TABLE II E F F E C T OF U R E A CONCENTRATION ON VISCOSITY AND OPTICAL ROTATORY PROPERTIES OF MYOSIN
Urea(M)
f *lJ
'Jo~]tD
~c'*
o*** 2*** 4 8 IO I2
2.2 2.2 o.9 1.2 1.2 1.2
27§ 43 § 98 97 lO3 95
31o 290 227 225 226 226
* Room temperature (23 °) except for 12 M urea which was measured at 52°. k ** From Drude equation [~]~.= 22 __ 202 *** KCI concentration was 0. 5 M, all other cases o.12 M. § For NEM-treated myosin [~]D = 39 and 56 for o and 2 M urea, respectively. T h e equilibrium n a t u r e of t h e m y o s i n - u r e a s y s t e m is clearly shown t h r o u g h o p t i c a l r o t a t o r y studies. A d d i t i o n of solid u r e a to an aqueous m y o s i n solution to a final c o n c e n t r a t i o n of 2 M results in a t i m e - d e p e n d e n t change in o p t i c a l r o t a t i o n w h i c h ceases a f t e r a b o u t 500 min a t 25 ° (see Fig. 7). T h e final value of E~]D = - - 4 3 °. T h e a d d i t i o n of solid u r e a to a c o n c e n t r a t i o n of 8 M gives, on t h e o t h e r h a n d , an i n s t a n t a n e o u s change to ~ ] D = - - 9 7 ° where the specific r o t a t i o n r e m a i n s i n v a r i a n t . D i l u t i o n of this l a t t e r solution to 2 M w i t h respect to urea results in an i m m e d i a t e depression of l a e v o r o t a t i o n to [~]D = - - 4 4 ° a n d t h e solution develops a significant bluish haze c h a r a c t e r i s t i c of t h e light s c a t t e r i n g from v e r y large particles. A similar reversal in t h e o p t i c a l r o t a t o r y p r o p e r t i e s is o b s e r v e d in t e m p e r a t u r e studies. On raising t h e t e m p e r a t u r e of a solution of m y o s i n in 2 M urea, t h e specific l a e v o r o t a t i o n undergoes a b r o a d t r a n s i t i o n b e t w e e n 25 a n d 5 o°, leveling off a t E~]D------ - 9 5 ° a t 65 °. On cooling this solution, t h e specific l a e v o r o t a t i o n r e t u r n s to [ ~ n = - - 4 9 °. Over t h e range of u r e a c o n c e n t r a t i o n s where these a s s o c i a t i o n - d i s s o c i a t i o n phen o m e n a are occurring, p o l y d i s p e r s e p a t t e r n s are also o b s e r v e d in gel electrophoresis studies. Fig. 6 p r e s e n t s t y p i c a l p a t t e r n s at u r e a c o n c e n t r a t i o n s b e t w e e n 4 a n d IO M . I n c o n t r a s t to t h e single s h a r p b a n d o b s e r v e d in t h e 12 M u r e a s y s t e m , t h r e e r a t h e r diffuse b a n d s are p r e s e n t at these c o n c e n t r a t i o n s . The diffuse c h a r a c t e r of t h e s e p a r a t e b a n d s a n d t h e v a r i a t i o n in w i d t h a n d i n t e n s i t y w i t h increasing u r e a c o n c e n t r a t i o n are f u r t h e r evidence of an a s s o c i a t i o n - d i s s o c i a t i o n equilibrium which is u r e a d e p e n d e n t . I n considering these electrophoresis p a t t e r n s , it is well to r e m e m b e r t h e m a r k e d m o l e c u l a r sieve effect one observes in such gels. Biochim. Biophys. Acta, 49 (1961) 462-470
469
ELECTROPHORESIS OF MYOSIN IN UREA
t
4M
}"
..
8M
.
1
IOM
Fig. 6. Acrylamide gel electrophoresis at different urea concentrations. Horizontal a r r o w indicates direction of migration, vertical a r r o w s indicate origins. F o r conditions, see text.
It will be apparent from the information presented above that at least three reactions are involved when myosin is transferred into concentrated urea solutions. These reactions are (a) the unfolding of the individual polypeptide helices, (b) the dissociation of the 3-stranded structure, and (c) the association processes which favor the formation of large molecular weight aggregates. At any given urea concentration, a balance is obtained among the three processes. Since the unfolding reaction would not be expected to have the same temperature coefficient as the reactions involving dissociation and association, it was thought that kinetic studies at different temperatures would provide some insight into the sequence of these basic mechanisms. Time-dependent changes in the optical rotation, viscosity, and adenosine triphosphatase activity in 2 M urea solutions are presented in Fig. 7 for temperatures of 7 and 25% The kinetics of these changes have not been analyzed formally. However, it can be seen from Table I I I that the half-life periods for the three properties are essentially the same in 2 M urea at each temperature. On the other hand, in 4 M urea the rates are strikingly different. The rates of change of adenosine triphosphatase activity and optical rotation which are reflecting configurational alterations in the
~
4
~
o
2
.
150 -~.. 140 r-I
130
- / "
/
~....e-- - - - - ' - ' - - - ~
iio
i 500
I,
I000 TIME (Minutes}
I
t// 1500
--
I 5000
Fig. 7. Time dependence of changes in optical rotation, viscosity and adenosine t r i p h o s p h a t a s e activity in 2 M urea at 7 °, • ; at 25 °, O. F o r o t h e r conditions, see text.
Biochirn. Biophys. Acta, 49 (1961) 462-47 o
47 °
P. A. SMALL, W. F. H A R R I N G T O N ,
W. W. KIELLEY
chains, are much more pronounced than the rate of change of viscosity which measures interaction and association, suggesting that the unfolding reaction precedes the processes of aggregation. TABLE KINETICS O F
III
AD]~NOSINE TRIPHOSPHATASE, OPTICAL ROTATORY AND
VISCOSITY CHANGES OF MYOSIN IN AQUEOUS UREA
Hall lives(miD) Urea
COq$C•.
70
25°
(M)
A TPase
/:¢]406.5
rJsp/C
A TPase
:'~ 7406.5
rlsp/C
2
iooo
117o
124o
35
25
25
4
~
1
~2o
~2ooo
<
io
~ioo
CONCLUSION
While the characteristics of these unfolding-association-dissociation reactions observed in the lower urea concentrations are of interest, the primary objective of the work reported here was to examine the electrochemical properties of the subunits of myosin. It is evident that complete dissociation into the individual polypeptide chains occurs only at or near a concentration of 12 M urea (at 40°). Under these conditions the subunits sediment in the ultracentrifuge as a single weight class and are monodisperse electrochemically, ACKNOWLEDGEMENT
The first author is a Special Research Fellow of the National Institutes of Health. REFERENCES 1 W . W . K I E L L E y AND W . F . HARRINGTON, Biochim. Biophys. Acta, 41 (196o) 4Ol. M. GELLERT AND S. W . ENGLANDER, i n preparation. 3 W . W . KIELLEY, H . M. KALCKAR AND L. B . :BRADLEY, J. Biol. Chem., 2 1 9 (1956) 95. 4 C. M. KAY, Biochim. Biophys. Acta, 38 (196o) 4 2 0 . 5 S. RAYMOND AND L. WEINTRAUB, Science, 1 3 o (1959) 711. s S. RAYMOND AND Y . WANG, Cyanogum 41 Gelling Agent as an Electrophoresis Medium /or Hemoglobin, American Cyanamide C o m p a n y , N . Y . 7 0 . SMITHIES, Advances in Protein Chem., V o l . 14, Academic Press, N . Y . , 1 9 5 9 , p. 65. 8 0 . SNELLMAN AND Z . ERDOS, Biochim. Biophys. Acta, 2 (1948) 65 o. t~ L . MANDELKERN AND l ~. J . FLORY, J. chim. phys., 2 0 (1952) 2 1 2 .
Biochim. Biophys. Acta, 4 9 (1961) 4 6 2 - 4 7 °
R. E. WINNICK, H. LIS, T. WINNICK ACKNOWLEDGEMENTS
We thank Dr. MONTAVU of Hoffman-La-Roche, Basel (Switzerland), for a supply of 5-fluorouracil, and Professor E. D. BERGMANN Of the Hebrew University, Jerusalem, for a sample of 5-fluorotryptophan. REFERENCES 1 G. V. CAUSE AND M. G. BRAZHNIKOVA, Lancet, (1944) 715 . 2 R. SCHWYZER, Record Chem. -Progr. (Kresge-Hooker Sci. Lib.), 20 (1959) 147. 3 j . M. BARRY AND E. ICHIHARA, Nature, 181 (1958) 1274. 4 j. L. STOKES AND C. R. WOODWARD, J. Bacteriol., 46 (1943) 83. s B. D. DAvIs AND E. S. MINGIOLI, J. Bacteriol., 60 (195o) 17. s E. KATZ, J. Biol. Chem., 235 (196o) lO9O. 7 W. D. MCELROY AND H. B. GLASS, A symposium on amino acid metabolism, J o h n s H o p k i n s U n i v e r s i t y Press, Baltimore, 1955, p. 277. s A. MEISTER, Biochemistry ol the amino acids, Academic Press, New York, 1957, C h a p t e r 4. 9 L. GORINI AND W. K. MAAS, Biochim. Biophys. Acta, 25 (1957) 2o8. 10 F. ZILLIKEI~!,Federation Proc., 18 (1959) 966. 11 V. V. KONINGSBERGER, C. O. VAN DER GRINTEN AND J. T. G. OVERBEEK, Biochim. Biophys. Acta, 26 (1957) 483 • 12 j . M. KIRK, Biochim. Biophys. Acta, 42 (196o) 167. 13 D. H. STRUMEYER AND K. BLOCH, J . Biol. Chem., 235 (196o) PC 27. 14 H. M. BATES AND F. LIPMANN, J. Biol. Chem., 235 (196o) PC 22. is E. ITo AND J. L. STROMINGER, dr. Biol. Chem., 235 (196o) PC 5. is :R. HANCOCK, Bioehim. Biophys. Acta, 37 (196o) 4717 I. UEMUZA, I~. SUYAMA AND C. J. CHERN, J . Osaka City Med. Center, 9 (196o) 1321, 2643, 2861.
Biochim. Biophys. Acta, 49 (1961) 451-462
THE ELECTROPHORETIC
HOMOGENEITY
OF T H E M Y O S I N S U B U N I T S PARKER
A. SMALL*, W I L L I A M F. H A R R I N G T O N * * AND W. W A Y N E K I E L L E Y
Laboratory o] Cellular Physiology and Metabolism, National Heart Institute, National Institutes oI Health, Bethesda, Md. (U.S.A .) Received N o v e m b e r 3rd, 196o)
SUMMARY
Gel electrophoresis of myosin in concentrated urea solutions demonstrates that the dissociated chains of myosin migrate as a monodisperse electrochemical species. At lower urea concentrations a complex dissociation-association system is obtained. Sedimentation, viscosity, optical rotation and gel electrophoresis studies under the latter conditions indicate the equilibrium nature of the system. Abbreviations: D M A P N , 2-dimethylaminopropionitrile; NEM, N-ethylmaleimide; Tris, tris(hydroxymethyl) aminomethane. * At p r e s e n t w i t h the W r i g h t - F l e m i n g I n s t i t u t e , St. M a r y ' s H o s p i t a l Medical School, Paddington, L o n d o n (Great Britain). ** P r e s e n t address: McCollum P r a t t I n s t i t u t e , J o h n s H o p k i n s University, Baltimore, Md. (U.S.A.).
Biochim. Biophys. ,4cta, 40 (1961) 462 -47 o
ELECTROPHORESIS OF MYOSIN IN UREA
463
INTRODUCTION
In a recent report t evidence was presented demonstrating that the myosin molecule is dissociated in aqueous 5 M guanidine. HC1 into three polypeptide chains of similar, if not identical, mass. The present study is concerned with the possible identity of the three polypeptide subunits. As a first approach to this problem, myosin-urea systems have been subjected to electrophoresis, utilizing an acrylamide gel (cyanogum No. 41 ) as matrix. As we will demonstrate below, urea concentrations of the order of 12 M are required to maintain complete dissociation of the individual chains of myosin. Under these conditions the polypeptide chains migrate as a single relatively sharp band in gel electrophoresis experiments over the p H range 7.2-9.5. I t thus appears that the subunit chains of myosin are electrochemically identical.
Myosin
MATERIALS AND METHODS
The method of preparation of myosin for these studies has been fully described elsewhere 1.
Experimental methods Myosin concentrations were determined spectrophotometrically assuming -279~I%_-5.60, a value recently established b y GELLERT AND ENGLANDER~ from careful dry weight measurements*.
Sedimentation studies Sedimentation was observed in a Spinco Model E Ultracentrifuge operating at 59,780 rev./min. Temperature control was achieved through the RTIC unit of the instrument. All runs were made in analytical cells equipped with Kel-F. Centerpieces. Sedimentation coefficients were corrected to a standard state of water at 20 ° assuming negligible binding of urea b y the protein (see ref. I for discussion); viscosities and densities of the various aqueous urea solvent systems were measured at the temperature of the ultracentrifuge runs ( + I°). In the calculation of s20,w values, a partial specific volume, ~ = 0.720 ml/g, was assumed. This was the value determined for myosin in 5 M guanidine.HC1 (see ref. I) and is consistent with the observations of KAY4 who showed t h a t the partial specific volume of a number of proteins decreases about 1 % when they are transferred to concentrated aqueous urea solvent.
Gel electrophoresis A modification of the gel electrophoresis method of RAYMOND5,6 was employed utilizing gels of acrylamide. These were formed in the presence of a suitable buffer system and the appropriate concentration of urea. The mixture of acrylamide and N,N' bismethyleneacrylamide monomer and the DMAPN were generously donated b y the American Cyanamide Company and are normally available under the trade names of Cyanogum No. 41 monomer and DMAPN catalyst, respectively. 3 g of * I n a n e a r l i e r p u b l i c a t i o n f r o m t h i s l a b o r a t o r y I t h e 2nd v i r i a l coefficient for m y o s i n i n 0. 5 M KC1 w a s g i v e n as 0.9o" IO-¢ mole m l / g z. T h i s n e w v a l u e for a b s o r b a n c y a t 279 m # ( c o n c n . = i %) c h a n g e s t h e v a l u e for t h e 2rid v i r i a l coefficient t o o.75" IO-e mol e m l / g I b u t does not, of course, a l t e r t h e i n f i n i t e d i l u t i o n v a l u e for t h e m o l e c u l a r w e i g h t (619,ooo). F o r t h e s a m e r e a s o n our p r e v i o u s l y p u b l i s h e d figure for t h e SH c o n t e n t of m y o s i n b e c o m e s 7.4 m o l e s / l ° s g.
Biochim. Biophys. Acta, 49 (1961) 462-47 o
464
P. A. S M A L L , W. F. H A R R I N G T O N ,
W. W. KIELLEY
monomer, the appropriate weight of urea and the volume of buffer solution noted in Table I, were diluted to a volume of 80 ml with water. After filtering the solution, 0.32 ml DMAPN catalyst and o.32 ml of a freshly prepared IO % ammonium persulfate solution were added (in that order) to initiate polymerization. Solutions were stirred gently to achieve mixing without aeration, then transferred to a lucite electrophoresis t r a y (25 × 4 × 0.5 cm) and covered. At room temperature, gelation required 30 to 12o min; at 40-50 ° (the temperature of the electrophoresis runs in 12 M urea system) gelation occurred in 3-30 min. TABLE
I
BUFFER SYSTEMS FOR GEL ELECTROPHORESIS pH
Gel (final v o l u m e 8 0 ml) Bridge (final v o l u m e 5 0 0 m l )
Stock reagents
7.z
8.6
9.5
2.o M Tris i.o M citric acid
2. 7 m l 2.0 m l
3.04 ml 0.40 ml
5.0 ml 0.05 ml
0. 5 M b o r i c a c i d 2. 5 M N a O H
5° ml o.I ml
3° 2. 4
ml ml
3° 4
ml ml
In general, the procedure of SMITHIES7 was followed in making electrophoresis runs. The protein, of concentration 5-25 mg/ml, was applied to a strip of W h a t m a n No. I filter paper which was then inserted into the gel (prior addition of urea to the myosin solutions was found to be unnecessary). A few drops of mineral oil were layered over the acrylamide matrix and the t r a y was covered with Saran Wrap to prevent evaporation of moisture during electrophoresis. The bridge buffer systems were made according to Table I. A voltage drop of 3-6 V/cm was sufficient to achieve an adequate migration rate. After completion of a run, electrophoretic patterns were developed b y placing the gel in a 1 % solution of Amido Black lOt3 dye (Hartman Leddon Company, Philadelphia) in methanol-water-acetic acid (5:5 : I, v/v) for 5-1o rain. Excess free dye was removed by washing the gel in m a n y changes of the same solvent (5:5:1) over a period of 4-5 days. RESULTS AND DISCUSSION
The dissociation of the myosin subunits in urea S N E L L M A N AND ERDOSs have demonstrated that myosin in aqueous 6 M urea solvent exhibits a complex sedimentation pattern. This is in contrast to sedimentation studies on myosin in aqueous 5 M guanidine. HC1 where the protein material sediments as a single weight class at all protein concentrations as demonstrated in Fig. I. A small, slow moving component, magnified b y the JOHNSTON-OGSTON affect, is apparent in the double sector cell experiment (6.1 mg protein/ml) in Fig. I. However, experience in this laboratory indicates that this material is a contaminant and can be considerably reduced b y repeating the ammonium sulfate fractionation in the preparation of myosin. All of our previous work on the dissociation of myosin into its component subunit chains was effected in 5 M guanidine. HC1, which is obviously unsuited for electrophoretic experiments. Consequently, a number of sedimentation B i o c h i m . B i o p h y s . A c t a , 4 9 (1961) 4 6 2 - 4 7 °
ELECTROPHORESIS OF MYOSIN IN UREA
465
Fig. I. Velocity s e d i m e n t a t i o n of m y o s i n in 5 M guanidine.HC1. The 2 and 4 m g / m l samples were t r e a t e d w i t h N E M and centrifuged at 59,78o r e v . / m i n for 179 a n d 189 rain, respectively; t h e 6.1 m g ] m l s a m p l e w a s u n t r e a t e d a n d centrifuged in a double sector cell at 5o,74 o r e v . / m i n for 448 m i n ; the 12. I m g / m l s a m p l e was treated with N E M and centrifuged f r o m a s y n t h e t i c b o u n d a r y ; the picture was t a k e n 128 rain after the centrifuge was b r o u g h t to 59,780 r e v . / m i n following I2o min at 12,34o.
experiments on various solutions of myosin in aqueous urea were performed in order to establish conditions for complete dissociation of the native molecule in this solvent*. Fig. 2 presents ultracentrifuge patterns obtained at various urea concentrations where it m a y be seen that complete dissociation of the myosin molecule into the slowest moving ( s z o , w ~ 1-2 S) peak is achieved only at urea concentrations of the order of 12 M (40°). The myosin samples employed in the experiments summarized in Fig. 2 were pretreated with NEM to prevent disulfide bridging among the dissociated chains 1. However, similar results were obtained when native myosin was added directly to either concentrated aqueous urea solvents or aqueous urea in the presence of o.I M fl-mercaptoethanol. In these experiments the ionic strength of the solvent system was
Fig. 2. Velocity s e d i m e n t a t i o n of m y o s i n as a f u n c t i o n of urea concentration. All p r o t e i n concent r a t i o n s 8 m g / m l ; KC1 concentration, 4-o.12 M, except the 2 M urea e x p e r i m e n t where 0.27 M KC1 was present. T e m p e r a t u r e , 2o-25 ° except for the 12 M urea e x p e r i m e n t (43% F o r o t h e r conditions, see text. * We are i n d e b t e d to Drs. J. T. EDSALL and D. ]3. WETI_AtIFER for informing us of their sedim e n t a t i o n w o r k on m y o s i n in 8 M urea solutions prior to publication.
Biochim. Biophys. Acta, 49 (1961) 462-470
466
P . A . SMALL, W. F. HARRINGTON, W. W. KIELLEY
critical. Thus, when myosin in IO M urea-o.I2 M KC1 solvent was examined in the ultracentrifuge, most of the protein sedimented as a slow moving boundary with sz0, w ---- 1.7 S (at a concentration of 8. 3 mg/ml) as shown in Fig. 2. On the other hand, myosin in IO M urea and the electrophoresis buffer system (pH 8.2) is predominantly undissociated. The sedimentation versus concentration dependence of myosin in various urea concentrations is shown in Fig. 4. In 12 M urea solvent where a single sedimenting boundary is observed (Fig. 2) the concentration dependence of sedimentation is much lower than that found for the major peak at lower urea concentrations. A plot of I/S versus C is linear and m a y be fitted b y the equation: I
I -
s
sO
(i + kc)
where c is the concentration in g/ml, k ---- 178 ml/g and S o ~- 3.51 S, the value of szo, w at infinite dilution. Assuming dissociation of the myosin macromolecule into random polypeptide chains of a single weight class in 12 M urea, it was of interest to estimate the chain weight. A molecular weight oi 18o,ooo was obtained from the FLORY-MANDELKERN equation 9, and the parameters s20' w ~ 3.51 S (Fig. 4) and E~] ---- 1.17 dl/g (Fig. 5). This value should be compared to a value of 197,ooo found in 5 M guanidine. HC1 (see ref. I) and supports our view that the chains are completely dissociated in 12 M urea.
Electrophoretic properties of the dissociated chains o/myosin Electrophoresis patterns of myosin in 12 M urea at various apparent p H values are shown in Fig. 3, demonstrating that a monodisperse migrating band was obtained under these solvent conditions over the p H range 7.2-9.5. At p H 8.6 and pH 9.5 the band had moved over essentially the length of the cyanogum gel with no indication of splitting. At p H 7.2 the migration rate was extremely slow, and in the time employed (48 h) for this study, diffusion had widened the band somewhat. At p H 9.5 electrophoresis for shorter time periods (5 h) yielded a single sharp band in contrast to the somewhat diffuse band depicted in Fig. 3 at this pH.
Fig. 3. Acrylamide gel electrophoresis of m y o s i n in 12 M urea a t different a p p a r e n t p H values. H o r i z o n t a l a r r o w indicates direction of migration, vertical arrows indicate origins. F o r conditions, see text.
The mechanism o/the dissociation o[ myosin in urea The complex behavior patterns of myosin in the presence of urea concentrations lower than 12 M is evident in Fig. 2. Several sedimenting components are present B i o c h i m . B i o p h y s . A c t a , 49 (i96I) 462-47 °
467
E L E C T R O P H O R E S I S OF MYOSIN IN U R E A
in any given urea concentration. A slow moving component is evident at urea concentrations of 2-8 M. This may be the contaminant referred to earlier. The sedimentation versus concentration dependence of the major peak (see Fig. 2) at various urea concentrations is presented in Fig. 4. In the 2 and 4 M urea systems it is apparent that very large molecular weight aggregates exist in the measurable concentration range of protein exhibiting infinite dilution values of S~o, w greater than 20 S. Higher urea concentrations favor the dispersal of these aggregates into the monomeric polypeptide chains. Viscosity measurements on the various urea solutions also emphasize the aggregation phenomena. Fig. 5 presents a system of reduced viscosity versus concentration plots at various urea concentrations. In general, data were obtained 24-48 h after the addition of urea or after the outflow times had reached constant values. Measured viscosities were essentially unchanged by the absence or presence of fl-mercaptoethanol. The extreme slope of the ~tsp/C versus C plot and the high values of ~sp/C at finite protein concentrations in 2 M urea is evidence of the massive concentrationdependent association in this solvent. One aspect of Fig. 5 is particularly striking. This is the apparent discontinuity in the sequence of the reduced viscosity versus concentration plots at 4 M urea where ~tsp/C versus C curve abruptly falls to comparatively low values. Nevertheless, the slope of the plot in 4 M urea suggests the presence of concentration-dependent aggregate, a view which is supported by the magnitude of s20,w of the major component observed in sedimentation studies (see Fig. 4). 7.0 6.5
20
x~
I
I
I
I
r
I
I
6.O
5.5 5.0
16
4.5
t4
12
~
4,0
~
3,5
o
4U
~'/"
3.(1 8 6 4
2 0
I
I
I
2
I' 3
I
I
I
I
I
4
5
6
7
S
PROTEIN CONCENTRATION (m~l/ml)
Fig. 4. C o n c e n t r a t i o n d e p e n d e n c e of s e d i m e n t a t i o n c o e f f i c i e n t s , s 20, w. a s a f u n c t i o n of u r e a c o n c e n t r a t i o n . 2 M u r e a , O; 4 M u r e a , & ; 8 M urea, [] ; i o M u r e a , • ; 12 M u r e a , ~ . T h e d a s h l i n e i n d i c a t e s t h e b e h a v i o r of n a t i v e m y o s i n in o. 5 M IZC1. F o r o t h e r c o n d i t i o n s , see t e x t .
0
0.1
0.2
0.3
0.4
o.5
0.6
0.7
0.8
PROTEIN CONCENTRATION ( g / l O O m | )
Fig. 5. R e d u c e d v i s c o s i t y v s . p r o t e i n c o n c e n t r a tion at various urea concentrations. 2 M urea, O; 4 M u r e a , &; 8 Murea, []; ioMurea, •; 12 M u r e a , <~. D a s h l i n e i n d i c a t e s b e h a v i o r of n a t i v e m y o s i n i n o. 5 M KC1.
It is instructive to compare changes in the optical rotatory properties which are a reflection, primarily, of the unfolding of the individual chains, with the viscosity Biochim. Biophys. A cta, 49 (1961) 4 6 2 - 4 7 °
468
P.A.
SMALL,
W. F. H A R R I N G T O N ,
W. W. KIELLEY
a n d s e d i m e n t a t i o n p r o p e r t i e s which sense the gross size a n d shape of t h e m a c r o m o lecules. I n 2 M urea, t h e specific o p t i c a l r o t a t i o n levels off a t a value (see T a b l e II) which is a b o u t 25 ~o of t h e over-all r o t a t o r y change o b s e r v e d a t high u r e a c o n c e n t r a tions. Y e t a t 4 M u r e a t h e m y o s i n chains are a b o u t 9 ° °/o u n f o l d e d as r e v e a l e d b y t h e values of [~]D a n d 2e, t h e D r u d e p a r a m e t e r , in T a b l e I I . Thus, it would seem t h a t in 2 M u r e a t h e m y o s i n molecules r e t a i n a considerable degree of r i g i d i t y which is conferred b y t h e presence of t h e high s-helical c o n t e n t , whereas a t 4 M u r e a t h e individual chains are e s s e n t i a l l y u n f o l d e d leading to a collapse of t h e rod-like s t r u c t u r e a n d t h e s u d d e n decline in r e d u c e d viscosity o b s e r v e d in Fig. 5. TABLE II E F F E C T OF U R E A CONCENTRATION ON VISCOSITY AND OPTICAL ROTATORY PROPERTIES OF MYOSIN
Urea(M)
f *lJ
'Jo~]tD
~c'*
o*** 2*** 4 8 IO I2
2.2 2.2 o.9 1.2 1.2 1.2
27§ 43 § 98 97 lO3 95
31o 290 227 225 226 226
* Room temperature (23 °) except for 12 M urea which was measured at 52°. k ** From Drude equation [~]~.= 22 __ 202 *** KCI concentration was 0. 5 M, all other cases o.12 M. § For NEM-treated myosin [~]D = 39 and 56 for o and 2 M urea, respectively. T h e equilibrium n a t u r e of t h e m y o s i n - u r e a s y s t e m is clearly shown t h r o u g h o p t i c a l r o t a t o r y studies. A d d i t i o n of solid u r e a to an aqueous m y o s i n solution to a final c o n c e n t r a t i o n of 2 M results in a t i m e - d e p e n d e n t change in o p t i c a l r o t a t i o n w h i c h ceases a f t e r a b o u t 500 min a t 25 ° (see Fig. 7). T h e final value of E~]D = - - 4 3 °. T h e a d d i t i o n of solid u r e a to a c o n c e n t r a t i o n of 8 M gives, on t h e o t h e r h a n d , an i n s t a n t a n e o u s change to ~ ] D = - - 9 7 ° where the specific r o t a t i o n r e m a i n s i n v a r i a n t . D i l u t i o n of this l a t t e r solution to 2 M w i t h respect to urea results in an i m m e d i a t e depression of l a e v o r o t a t i o n to [~]D = - - 4 4 ° a n d t h e solution develops a significant bluish haze c h a r a c t e r i s t i c of t h e light s c a t t e r i n g from v e r y large particles. A similar reversal in t h e o p t i c a l r o t a t o r y p r o p e r t i e s is o b s e r v e d in t e m p e r a t u r e studies. On raising t h e t e m p e r a t u r e of a solution of m y o s i n in 2 M urea, t h e specific l a e v o r o t a t i o n undergoes a b r o a d t r a n s i t i o n b e t w e e n 25 a n d 5 o°, leveling off a t E~]D------ - 9 5 ° a t 65 °. On cooling this solution, t h e specific l a e v o r o t a t i o n r e t u r n s to [ ~ n = - - 4 9 °. Over t h e range of u r e a c o n c e n t r a t i o n s where these a s s o c i a t i o n - d i s s o c i a t i o n phen o m e n a are occurring, p o l y d i s p e r s e p a t t e r n s are also o b s e r v e d in gel electrophoresis studies. Fig. 6 p r e s e n t s t y p i c a l p a t t e r n s at u r e a c o n c e n t r a t i o n s b e t w e e n 4 a n d IO M . I n c o n t r a s t to t h e single s h a r p b a n d o b s e r v e d in t h e 12 M u r e a s y s t e m , t h r e e r a t h e r diffuse b a n d s are p r e s e n t at these c o n c e n t r a t i o n s . The diffuse c h a r a c t e r of t h e s e p a r a t e b a n d s a n d t h e v a r i a t i o n in w i d t h a n d i n t e n s i t y w i t h increasing u r e a c o n c e n t r a t i o n are f u r t h e r evidence of an a s s o c i a t i o n - d i s s o c i a t i o n equilibrium which is u r e a d e p e n d e n t . I n considering these electrophoresis p a t t e r n s , it is well to r e m e m b e r t h e m a r k e d m o l e c u l a r sieve effect one observes in such gels. Biochim. Biophys. Acta, 49 (1961) 462-470
469
ELECTROPHORESIS OF MYOSIN IN UREA
t
4M
}"
..
8M
.
1
IOM
Fig. 6. Acrylamide gel electrophoresis at different urea concentrations. Horizontal a r r o w indicates direction of migration, vertical a r r o w s indicate origins. F o r conditions, see text.
It will be apparent from the information presented above that at least three reactions are involved when myosin is transferred into concentrated urea solutions. These reactions are (a) the unfolding of the individual polypeptide helices, (b) the dissociation of the 3-stranded structure, and (c) the association processes which favor the formation of large molecular weight aggregates. At any given urea concentration, a balance is obtained among the three processes. Since the unfolding reaction would not be expected to have the same temperature coefficient as the reactions involving dissociation and association, it was thought that kinetic studies at different temperatures would provide some insight into the sequence of these basic mechanisms. Time-dependent changes in the optical rotation, viscosity, and adenosine triphosphatase activity in 2 M urea solutions are presented in Fig. 7 for temperatures of 7 and 25% The kinetics of these changes have not been analyzed formally. However, it can be seen from Table I I I that the half-life periods for the three properties are essentially the same in 2 M urea at each temperature. On the other hand, in 4 M urea the rates are strikingly different. The rates of change of adenosine triphosphatase activity and optical rotation which are reflecting configurational alterations in the
~
4
~
o
2
.
150 -~.. 140 r-I
130
- / "
/
~....e-- - - - - ' - ' - - - ~
iio
i 500
I,
I000 TIME (Minutes}
I
t// 1500
--
I 5000
Fig. 7. Time dependence of changes in optical rotation, viscosity and adenosine t r i p h o s p h a t a s e activity in 2 M urea at 7 °, • ; at 25 °, O. F o r o t h e r conditions, see text.
Biochirn. Biophys. Acta, 49 (1961) 462-47 o
47 °
P. A. SMALL, W. F. H A R R I N G T O N ,
W. W. KIELLEY
chains, are much more pronounced than the rate of change of viscosity which measures interaction and association, suggesting that the unfolding reaction precedes the processes of aggregation. TABLE KINETICS O F
III
AD]~NOSINE TRIPHOSPHATASE, OPTICAL ROTATORY AND
VISCOSITY CHANGES OF MYOSIN IN AQUEOUS UREA
Hall lives(miD) Urea
COq$C•.
70
25°
(M)
A TPase
/:¢]406.5
rJsp/C
A TPase
:'~ 7406.5
rlsp/C
2
iooo
117o
124o
35
25
25
4
~
1
~2o
~2ooo
<
io
~ioo
CONCLUSION
While the characteristics of these unfolding-association-dissociation reactions observed in the lower urea concentrations are of interest, the primary objective of the work reported here was to examine the electrochemical properties of the subunits of myosin. It is evident that complete dissociation into the individual polypeptide chains occurs only at or near a concentration of 12 M urea (at 40°). Under these conditions the subunits sediment in the ultracentrifuge as a single weight class and are monodisperse electrochemically, ACKNOWLEDGEMENT
The first author is a Special Research Fellow of the National Institutes of Health. REFERENCES 1 W . W . K I E L L E y AND W . F . HARRINGTON, Biochim. Biophys. Acta, 41 (196o) 4Ol. M. GELLERT AND S. W . ENGLANDER, i n preparation. 3 W . W . KIELLEY, H . M. KALCKAR AND L. B . :BRADLEY, J. Biol. Chem., 2 1 9 (1956) 95. 4 C. M. KAY, Biochim. Biophys. Acta, 38 (196o) 4 2 0 . 5 S. RAYMOND AND L. WEINTRAUB, Science, 1 3 o (1959) 711. s S. RAYMOND AND Y . WANG, Cyanogum 41 Gelling Agent as an Electrophoresis Medium /or Hemoglobin, American Cyanamide C o m p a n y , N . Y . 7 0 . SMITHIES, Advances in Protein Chem., V o l . 14, Academic Press, N . Y . , 1 9 5 9 , p. 65. 8 0 . SNELLMAN AND Z . ERDOS, Biochim. Biophys. Acta, 2 (1948) 65 o. t~ L . MANDELKERN AND l ~. J . FLORY, J. chim. phys., 2 0 (1952) 2 1 2 .
Biochim. Biophys. Acta, 4 9 (1961) 4 6 2 - 4 7 °